The goals of Project 3 are to develop and apply methods for expanding transplanted human cells in a pediatric nonhuman primate model that can be readily monitored and tracked using in vivo imaging. To increase the quantity of cells available for transplant we will obtain hematopoietic cells differentiated from human embryonic stem cells (hESC) that will be expanded by co-culture with human endothelial progenitor cells (EPC) based on protocols developed in Project 1. Transplant of early hematopoietic cells, rather than mature cells, may be central to the success of cell-based therapies. To improve the levels of engraftment and increase the quantity of cells post-transplant, we will transduce cells with a drug resistance gene. Therefore, one of the goals of these studies is to use differentiated hESC in a side-by-side comparison with adult human peripheral blood stem cells (PBSC). These investigations will allow us to focus on methods to expand stem and progenitor cells in infant monkeys which could have significant clinical utility. The transplant of cells differentiated from hESC in this pediatric nonhuman primate model will also address concerns related to the in vivo transplantation of these cells. In addition, our prior studies have shown that we can monitor gene expression in fetal and infant monkeys using reporter genes, microPET, and optical imaging. Based on these findings we will use similar protocols to inject, image, and track transplanted human stem and progenitor cells expanded postnatally in this model. Preliminary studies have shown that labeling of cells with radioactive copper does not harm the cells and permits cell tracking for several days. While these studies provide a crucial step towards developing techniques for human cell trafficking, safe, sensitive, and efficient methods for monitoring cells longitudinally and over time that can be translated to humans are needed. These investigations will allow us to focus on semi-quantitative methods for cell imaging, and the use of selection and in vivo imaging protocols simultaneously in infant nonhuman primates. If imaging technologies with sufficient sensitivity to detect small quantities of cells can be used, this would provide a very powerful tool for assessing the safety and efficiency of cell-based therapies in humans. California National Primate Research Center, University of California, Davis CA PHS 398 (Rev. 09/04) Page 166 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): Tarantal, Alice F (Project 3) KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Tarantal, Alice F. ATARANTAL CNPRC, UC Davis Project Leader Cherry, Simon R. scherry UC Davis Co-Investigator OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Firpo, Meri T. University of Minnesota Consultant Gerson, Stanton L. Case Western Reserve University Consultant Weinberg, Kenneth I. CHLA Consultant Human Embryonic Stem Cells D No [unreadable]3 Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell llne(s) from the following list: http://stemcells.nih.gov/reqistrv/index.asp. Usecontinuation pages asneeded, If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used. Cell Line UC06 Disclosure Permission Statement. Applicable toSBIR/STTR Only. See instructions, d Yes O No PHS 398 (Rev. 09/04) Page 167 Form Page 2-continued Number the following pages consecutively throughoutthe application. Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Tarantal, Alice F. (Project 3) DETAILED BUDGET FOR INITIAL BUDGET PERIOD FROM THROUGH DIRECT COSTS ONLY 09/01/05 8/31/06 PERSONNEL (Applicant organization only) % DOLLAR AMOUNT REQUESTED (omit cents) TYPE EFFORT INST. ROLE ON APPT. ON BASE SALARY FRINGE NAME PROJECT (months) PROJ. SALARY REQUESTED BENEFITS TOTAL Principal Alice F. Tarantal Investigator 12 10.0 137,475 13,748 2,475 16,223 Travis S. Burns Techician 12 100.0 40,932 40,932 12,412 53,344 \ Simon R. Cherry Collaborator 12 3.0 158,860 4,766 858 5,624 SUBTOTALS 59,446 15,745 75,1911 CONSULTANT COSTS Drs. Firpo, Gerson, Weinberg 0 EQUIPMENT /Itemize} None 0 SUPPLIES (Itemize by category) Pheresis costs ($5,000), Supplies for preparation of cells for transplant, progenitor assays, PCR, antibodies, ELISAs, quantitative imaging experiments (cells and tissues), alkylating agent, Imaging costs 56,939 TRAVEL None PATIENT CARE COSTS INPATIENT OUTPATIENT ALTERATIONS AND RENOVATIONS (Itemize by category) OTHER EXPENSES (Itemize by category) Miscellaneous office and computer, service contracts, EH&S costs 1,000 CONSORTIUM/CONTRACTUAL COSTS DIRECT COSTS SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item 7a, Face Page) $ 133,130| CONSORTIUM/CONTRACTUAL COSTS FACILITIES AND ADMINISTRATIVE COSTS TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD $ 133,130| SBIR/STTR Only: FEE REQUESTED PHS 398 (Rev. 09/04) Page 168 Form Page 4